RESEARCH ARTICLE

ECOLOGY 2016 © The Authors, some rights reserved;

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Synergistic roles of climate warming and human the Advancement of Science. Distributed
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occupation in Patagonian megafaunal extinctions NonCommercial License 4.0 (CC BY-NC).

10.1126/sciadv.1501682

during the Last Deglaciation

Jessica L. Metcalf,1,2 Chris Turney,3* Ross Barnett,4,5* Fabiana Martin,6 Sarah C. Bray,1,7 Julia T. Vilstrup,5
Ludovic Orlando,5 Rodolfo Salas-Gismondi,8,9 Daniel Loponte,10 Matías Medina,11 Mariana De Nigris,12
Teresa Civalero,12 Pablo Marcelo Fernández,12 Alejandra Gasco,13 Victor Duran,13 Kevin L. Seymour,14
Clara Otaola,15 Adolfo Gil,15 Rafael Paunero,16 Francisco J. Prevosti,17 Corey J. A. Bradshaw,18 Jane C. Wheeler,19
Luis Borrero,20 Jeremy J. Austin,1 Alan Cooper1,4†

The causes of Late Pleistocene megafaunal extinctions (60,000 to 11,650 years ago, hereafter 60 to 11.65 ka) remain

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contentious, with major phases coinciding with both human arrival and climate change around the world. The
Americas provide a unique opportunity to disentangle these factors as human colonization took place over a
narrow time frame (~15 to 14.6 ka) but during contrasting temperature trends across each continent. Unfortunately,
limited data sets in South America have so far precluded detailed comparison. We analyze genetic and radiocarbon
data from 89 and 71 Patagonian megafaunal bones, respectively, more than doubling the high-quality Pleistocene
megafaunal radiocarbon data sets from the region. We identify a narrow megafaunal extinction phase 12,280 ± 110 years
ago, some 1 to 3 thousand years after initial human presence in the area. Although humans arrived immediately
prior to a cold phase, the Antarctic Cold Reversal stadial, megafaunal extinctions did not occur until the stadial
finished and the subsequent warming phase commenced some 1 to 3 thousand years later. The increased resolu-
tion provided by the Patagonian material reveals that the sequence of climate and extinction events in North and South
America were temporally inverted, but in both cases, megafaunal extinctions did not occur until human presence and
climate warming coincided. Overall, metapopulation processes involving subpopulation connectivity on a continental
scale appear to have been critical for megafaunal species survival of both climate change and human impacts.

INTRODUCTION
The loss of Late Pleistocene megafaunal diversity in South America was
among the greatest of any continent (52 genera, 83%), and Fell’s Cave
1
Australian Centre for Ancient DNA, School of Biological Sciences, University of Adelaide, Ade-
laide, South Australia 5005, Australia. 2Department of Ecology and Evolutionary Biology, Ramaley
Biology, University of Colorado, Boulder, CO 80309–0334, USA. 3Climate Change Research Cen-
tre, School of Biological, Earth, and Environmental Sciences, University of New South Wales,
Sydney, Australia. 4Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, Uni-
versity of Oxford, South Parks Road, Oxford OX1 3PS, UK. 5Centre for GeoGenetics, Natural
History Museum of Denmark, University of Copenhagen, Øster Voldgade 5–7, DK-1350 Copen-
hagen, Denmark. 6Centro de Estudios del Hombre Austral, Instituto de la Patagonia, UMAG,
Avenida Bulnes 01890, Punta Arenas, Chile. 7Acute Leukaemia Laboratory, Centre for Cancer
Biology, University of South Australia, Adelaide South Australia 5001, Australia. 8Institut des
Sciences de l’Evolution, Université de Montpellier, CNRS, IRD, EPHE, Montpellier 34095, France.
9
Departamento de Paleontologia de Vertebrados, Museo de Historia Natural, UNMSM, Avenida
Arenales 1256, Lima 14, Peru. 10Instituto Nacional de Antropología y Pensamiento Latino-
americano, C1426BJN Ciudad de Buenos Aires, Argentina. 11Área de Arqueología y Etnohistoria,
Centro de Estudios Históricos “Prof. Carlos S.A. Segreti,” Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Miguel C. del Corro 308, Córdoba (5000), Argentina. 12Instituto
Nacional de Antropología y Pensamiento Latinoamericano (INAPL), CONICET/UBA, 3 de Febrero
1370, C1426BJN Buenos Aires, Argentina. 13CONICET, Laboratorio de Paleoecología Humana,
Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, Mendoza, Argentina.
14
Department of Natural History, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario
M5S 2C6, Canada. 15CONICET-IANGLA Grupo Vincualdo San Rafael/ UTN-MHNSR, Parque Mar-
iano Moreno (5600), San Rafael, Mendoza, Argentina. 16Departamento Científico de Arqueo-
logía. Facultad de Ciencias Naturales y Museo, UNLP, Avenida Paseo del Bosque s/n (1900), La
Plata, Buenos Aires, Argentina. 17Centro Regional de Investigaciones Científicas y Transferencia
Tecnológica de La Rioja (CRILAR), Provincia de La Rioja, UNLaR, SEGEMAR, UNCa, CONICET,
Entre Ríos y Mendoza s/n, (5301), Anillaco, La Rioja, Argentina. 18School of Biological Sciences,
University of Adelaide, Adelaide, South Australia 5005, Australia. 19CONOPA, Instituto de
Investigación y Desarrollo de Camélidos Sudamericanos, Lima, Peru. 20CONICET-IMHICIHU,
Universidad de Buenos Aires. Saavedra 15, 5 (1083 ACA), Buenos Aires, Argentina.
*These authors contributed equally to this work.
†Corresponding author. Email: alan.cooper@adelaide.edu.au
in Patagonia was one of the first sites globally where archaeological
evidence of human hunting was associated with megafaunal remains
(1–3). Despite this, the timing and nature of the South American mega-
faunal extinctions and the evolutionary relationships of many taxa
remain poorly understood. Patagonia provides an ideal study area in
this respect because of the excellent preservation of Late Pleistocene fos-
sil remains compared to the rest of South America (4, 5), and the broad-
ly synchronous but antiphase climate changes to those in the Northern
Hemisphere (commonly referred to as the bipolar seesaw) (6, 7).
Following maximum glacial extent across the region 28.5 thousand
years ago (ka) (8), long-term warming from 18 ka to the Holocene was
interrupted between ~14.4 and 12.7 ka by a marked 1700-year decline
in temperature and glacial advance identified as the Antarctic Cold
Reversal (ACR) stadial (Fig. 1) (7, 9). Glacial conditions persisted
until ~12.6 ka, when rapid warming and retreat of the Patagonian
Ice Sheet led to the expansion of Nothofagus forest ~12.3 ka, with
peak Holocene warming attained by 11.4 ka (10, 11). Crucially, the first
records of human presence in southern Chile occur immediately prior
to the ACR stadial, at Monte Verde near Puerto Montt on the west-
ern edge of Patagonia, before ~14.6 ka (12, 13). The central plateau of
northern Patagonia was occupied before ~13.2 ka (14), with the first
confirmed occupation in southern Patagonia between ~13.2 and 12.9 ka,
and ~12.7 ka in Tierra del Fuego (14, 15). In contrast to the relatively
intense occupation of the central plateau, the first human occupations in
southern Patagonia appear relatively ephemeral, with the number of
sites and intensity of occupation increasing markedly from the early
Holocene (15, 16).

Metcalf et al. Sci. Adv. 2016; 2 : e1501682 17 June 2016 1 of 8

RESEARCH ARTICLE

−34

Greenland (NGRIP) δ18O, ‰

Holocene GS-1 GI-1 GS-2
−36
Greenland δ18O
−38

−40

−42

Summed probability
−44 0.004
North America
0.002

Extinction
Patagonia

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L. guanicoe (extinct)

L. gracilis

P. onca mesembrina

H. saldiasi

M. darwinii

S. populator

Arctotherium

MV H. sapiens

Holocene L. guanicoe

−32

−34
WAIS δ18O, ‰

West Antarctic δ18O

−36

−38

Antarctic
−40
Cold
Reversal (ACR)
9000 10,000 11,000 12,000 13,000 14,000 15,000 16,000 17,000 18,000
Years before pesent
Fig. 1. Extinction chronology of Pleistocene megafauna in southern Patagonia (9000 to 18,000 years ago), shown against West Antarctic Ice Sheet
Divide ice d18O record (bottom) and the timing of Antarctic Cold Reversal chronozone (blue column) (9). Calibrated radiocarbon (14C) ages of extinct
megafaunal remains from Patagonia [open circles with 1s uncertainty indicated by vertical whiskers] are shown with calculated Phase boundary start and
end (extinction) estimates (filled red squares) using the Southern Hemisphere calibration (SHCal13) curve (51) and OxCal 4.2 (26). The vertical gray bar
denotes the 1s range for megafaunal extinction in Patagonia. Calibrated 14C ages for the arrival of humans (with Monte Verde shown as “MV” with dashed
line for extended age range) and modern lineage of L. guanicoe are shown as open circles and red square. (Top) A summary of the Northern Hemisphere
climate and North American megafaunal extinctions is also presented. The Greenland d18O and isotope event stratigraphy are shown (GS, Greenland
Stadial; GI, Greenland Interstadial) (60), with the summed probabilities (1s) of the youngest 14C dates for the latest Pleistocene megafaunal extinctions in
North America (27), using the IntCal13 calibration curve (59). The tan and blue shading highlights the inverted timing of warm and cold intervals in the
Northern Hemisphere and Southern Hemisphere during the Last Termination. WAIS, West Antarctic Ice Sheet; NGRIP, North Greenland Ice-Core Project.

Metcalf et al. Sci. Adv. 2016; 2 : e1501682 17 June 2016 2 of 8

RESEARCH ARTICLE

To characterize the extinction of the Late Pleistocene Patagonian
megafauna, we sequenced mitochondrial DNA (mtDNA) from 89
megafaunal bone and teeth samples recovered from caves and rock
shelters, and generated new accelerator mass spectrometry radiocarbon
(AMS 14C) dates for a total of 71 bone, teeth, and coprolite samples
(tables S1 to S3). We then investigated whether the megafaunal extinc-
tions were associated with major events in records of climate change
and/or human occupation.
RESULTS
Phylogenetic relationships of Pleistocene megafauna
Phylogenetic analysis of the new genetic data (see the Supplementary
Materials) reveals that the Late Pleistocene megafaunal extinctions con-
the giant short-faced bear (Arctotherium sp.) (23), and the South Amer-
ican horse (Hippidion saldiasi) (24, 25), to compare extinction dates
with the arrival of both humans and the genetically distinct modern
lineage of L. guanicoe (Fig. 1). We used the Phase calibration function
with Outlier analysis in OxCal 4.2 to define the midpoint of the start and
end (estimated extinction) of the set of ages for each species (26), which
revealed that the megafaunal extinctions most likely occurred in a nar-
row time window spanning 12.2 to 12.5 thousand years (table S4). In-
dividual age calibrations of the youngest samples confirm that
extinction took place around this time (table S4). Using the C_Combine
function in OxCal, we find that the ages for the different species are
statistically indistinguishable, implying that extinction across Patagonia
was synchronous, with a mean weighted age of 12,280 ± 110 years
ago. By doubling the number of high-quality 14C-dated megafaunal fos-
sils for this region, we do not observe carnivore extinctions preceding

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tained several previously unknown events, including a distinct camelid
species, Lama gracilis (17), a previously unknown genetic clade of gua-
naco (Lama guanicoe) (Fig. 2), and a genetically distinct giant South
American jaguar subspecies, Panthera onca mesembrina (18). In con-
trast, the single mitochondrial haplotype recovered from ancient south-
ern Patagonian puma specimens (Puma concolor) appears closely related
to haplotypes surviving in South America at present.
Extinction of Pleistocene megafauna and associations
with human occupation and climate
We combined the newly dated specimens with previously published
14
C data of extinct megafauna, such as the large ground sloth (Mylodon
darwinii) (19, 20), the saber-toothed cat (Smilodon populator) (21, 22),
that of herbivores as recently suggested (5). Humans were clearly pres-
ent in Patagonia for at least 1000 years before the extinction event (Fig.
1 and table S5), and probably more than 2300 years if Monte Verde more
accurately provides a minimum estimate of the occupation of the wider
Patagonian area (13).
The prolonged temporal overlap between humans and megafauna
in Patagonia (from around 14.6 to 13.2 ka through to the extinction
events at 12.3 ka) corresponds closely in timing to the pronounced cold
conditions of the ACR stadial (Fig. 1). In contrast, the tight cluster of
Patagonian megafaunal extinctions occurs shortly after the start of the
rapid warming phase that followed the termination of the ACR. Previ-
ous studies of southern Patagonian lake sediments have suggested that
the post-ACR environment was arid and windy, with variable rainfall

Camelids Jaguar Puma

1.0/0.94
L. gracilis
0.99/0.90
100 Panthera leo
Panthera pardus
1.0/0.88 V. vicugna

Ancient Patagonian
1.0/1.0
Puma concolor
Patagonian
1.0/0.98
L. guanicoe
96
Modern
1.0/
100 Panthera
onca

1.0/
L. guanicoe

Ancient
P. onca mesembrina
1.0/1.0
Camelus

Fig. 2. Mitochondrial DNA phylogenies of Late Pleistocene Patagonian megafauna (red) relative to extant taxa (black). (A) Phylogenetic recon-
structions of ancient and modern camelid sequences revealed two groups of camelids: Lama gracilis and a distinct clade of L. guanicoe. (B) Pleis-
tocene Panthera onca were distinct from modern jaguars and represent the extinct subspecies P. onca mesembrina. (C) In contrast, sequences from
Pleistocene Puma concolor are closely related to modern haplotypes. All Pleistocene samples represent haplotypes that are extinct or unsampled in
modern populations.

Metcalf et al. Sci. Adv. 2016; 2 : e1501682 17 June 2016 3 of 8

RESEARCH ARTICLE
and potential summer droughts (11). By 12.3 ka, when the extinctions
occured, glacial ice was retreating and sufficient moisture was available
to support rapid expansion of Nothofagus forest, consistent with the
warming observed in the WAIS record (9).
DISCUSSION
The close association of the Patagonian megafaunal extinctions with
the warming phase following the ACR agrees with recent studies sug-
gesting that interstadials were a key driver of Holarctic megafaunal
extinctions through the Late Pleistocene (27). However, the reversed
temperature trends in North and South America during this period led
to a marked inversion in the sequence of events and timing (Fig. 1). In
North America, multiple megafaunal extinctions are inferred between
14.6 and 12.8 ka during the warm Greenland Interstadial 1, followed by
a relative hiatus during the cold Greenland Stadial 1, before the last ex-
tinctions after 11.6 ka in the early Holocene (27). The pattern in south-
ern Patagonia is temporally exactly inverted but also shows megafaunal
extinctions occurred during a warming phase and a hiatus during a sta-
dial (Fig. 1). In addition, the extinction process in North America
appears considerably more protracted, at least from our data, potentially
as a consequence of the hiatus during Greenland Stadial 1 in the middle
of the event.
The Patagonian camelid data also appear to reflect metapopulation
processes similar to those inferred from ancient DNA studies of several
Late Pleistocene Northern Hemisphere megafaunal taxa, including
cave bear, bison, and mammoth (27–29). Following the extinction of
Patagonian L. gracilis and the Late Pleistocene clade, or possibly sub-
species, of L. guanicoe around 12.3 ka, a genetically distinct northern
population of L. guanicoe moved into the region. Early Holocene came-
lid bone deposits from Cerro Casa del Piedra 7 in southern Patagonia
(fig. S1) provide a minimum age (10,551 ± 170 years ago) for the ap-
pearance of the replacement northern population (see the Supplemen-
tary Materials). Since the latter is ancestral to all modern guanaco
stocks, this metapopulation process also appears to have been respon-
sible for the species’ survival. The observation of similar rapid
continental-scale conspecific replacements in both the Holarctic and
Patagonia suggests that such metapopulation structures were common
(28, 30–32), reinforcing the hypothesis that they may have evolved to
provide resilience to the frequent and marked climatic shifts observed
throughout the Pleistocene (27).
Although the Patagonian genetic record has a more limited tem-
poral span than the Northern Hemisphere records previously reported
(27), the higher resolution afforded by our study reveals the interplay
between human impacts and climate change. The initial presence of
humans in the area during the ACR stadial conditions was appar-
ently insufficient to drive megafaunal extinctions, in direct contrast
to the Blitzkrieg model, which suggests that the naïveté of mega-
fauna to human hunting led to rapid extinction (33). However, human
presence, in combination with the rapid advance of forests and en-
vironmental changes associated with the ensuing warming phase,
appears to have led to the collapse of the megafaunal ecosystem with-
in a few hundred years. It is unclear whether rapid increases in human
population size were associated with the warming phase, although an-
cient DNA analyses indicate a very rapid demographic growth among
the founding American populations (34). The new genetic data are
consistent with the hypothesis that human disruption of megafaunal
Metcalf et al. Sci. Adv. 2016; 2 : e1501682 17 June 2016
metapopulation processes (for example, long distance dispersal and
rescue effects), along with increased hunting pressure associated
with decreased habitat range, allowed local population extinctions
initiated by environmental change to coalesce into a larger ecosystem-
wide alteration, potentially with minimal direct signs of human hunt-
ing (27).
MATERIALS AND METHODS
We collected 175 Late Pleistocene and Holocene megafaunal bone
and teeth fragments from museum collections in Argentina (La Plata
Museum, La Plata Zoo, Instituto de Ciencias Básicas, Universidad
Nacional de Cuyo, and Museo de Historia Natural de San Rafael), Chile
(Centro de Estudios del Hombre Austral, Instituto de la Patagonia,
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and Universidad de Magallanes), Perú (Departamento Científico de
Arqueología and Facultad de Ciencias Naturales y Museo), Netherlands
(Zoological Museum Amsterdam), Sweden (Museum of Natural History,
Malmo), and Russia (Moscow State University Zoological Museum) (tables
S1 and S2). Most of the specimens were originally collected from several
well-known caves in the Última Esperanza region of southern Patagonia,
including Cueva del Milodón, Cueva Lago Sofía 4, and Cueva del Medio.
Our sample collection also included megafauna subfossil material from
caves in the Patagonia regions of Tierra del Fuego at the southernmost
tip of Patagonia, the volcanic area of Pali Aike, and central Santa Cruz
(fig. S1). We also sampled Holocene camelid material from these sites in
Patagonia, as well as several other caves in the region (fig. S1). The Cerro
Casa de Piedra site in the Santa Cruz region of Patagonia contains
deposits of guanaco material spanning the early Holocene to recent
times. Finally, we included several camelid samples collected from Peru-
vian caves to examine which lineages of camelids were present outside of
Patagonia during the Pleistocene and the Holocene.
We sampled well-preserved megafaunal remains, primarily cor-
tical limb bone and tooth roots, using a Dremel drill and disposable
carborundum cutting discs. Samples of approximately 100 mg were
removed and transferred to the ancient DNA laboratory.
DNA extraction and sequencing of mtDNA
We primarily carried out ancient DNA laboratory procedures at the
Australian Centre for Ancient DNA (ACAD) in Adelaide, Australia,
although the felid specimens had previously been examined at the
Henry Wellcome Ancient Biomolecules Centre in Oxford, UK. We
followed standard ancient DNA protocols for DNA extraction and
polymerase chain reaction (PCR) setup in a physically isolated, clean
laboratory with positive air pressure, HEPA-filtered air flow and
ultraviolet(UV) lights, regular bleaching, and multiple controls for
all stages of the procedures (35). Before extraction, we attempted to
remove potential contaminants by removing the outer layer of each
sample using a Dremel drill and disposable carborundum cutting discs
and by exposing the sample to UV light. DNA was extracted using
either phenol-chloroform (36) or silica-based extraction methods (37).
We amplified a number of phylogenetically informative mito-
chondrial fragments (for example, control region, cytochrome b,
ATP8, and/or ND5) for each sample to confirm morphological iden-
tification (table S3). Due to the degraded nature of the samples, we
amplified multiple, short, overlapping DNA fragments. We detail
the PCR conditions for each group of taxa below (camelids, felids,
and bear).
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RESEARCH ARTICLE
Camelid samples. Six control region fragments and one cyto-
chrome b fragment were targeted for PCR using primers listed in table
S3. Two sets of multiplex PCRs were performed to amplify all seven
fragments (38). Multiplex PCRs were done in a final volume of 25 ml
using 1 to 2 U of Platinum Taq High-Fidelity DNA Polymerase and
1× buffer (Invitrogen), rabbit serum albumin (RSA) (2 mg/ml; Sigma),
2 mM MgSO4, 250 mM of each deoxynucleotide triphosphate (dNTP),
1 mM of each primer, and 1 to 4 ml of DNA. PCRs were run at 94°C for
1 min followed by 30 cycles (multiplex) or 50 cycles (singleplex) at 94°C
for 15 s, 55°C for 15 s, and 68°C for 30 s, and a final elongation step at
68°C for 10 min. A no-template control and a negative extraction con-
trol were included for approximately every four samples. The second
round of PCR was performed in singleplex reactions with 0.5 U of
HotMaster Taq, 1× buffer (HotMaster), 200 mM of each dNTP, bovine
serum albumin (BSA) (1 mg/ml), 1 mM of each primer, and a 100-fold
dilution of the first-round PCR product. PCR products were visualized
on a 4% tris-borate EDTA agarose gel. In rare cases in which a negative
control exhibited a band, the PCR was discarded and not used for
sequencing.
Felid samples. The general carnivore primers ATP8_1F/
ATP8_3R were initially used to identify the species, followed by more
species-specific overlapping primer pairs to amplify regions of the
Smilodon, puma, and jaguar samples (table S3). PCR amplifications
(25 ml of total volume) were done using Platinum Taq High-Fidelity
DNA Polymerase (1.25 U; Invitrogen), 1× buffer (Invitrogen), BSA
(0.2% w/v; Sigma), 2 mM MgSO4, 2′-deoxynucleotide 5′-triphosphate
mix (all 25 mM), 1 ml of an extract, and 1 mM each of forward and
reverse primers. A 2-min activation step at 94°C was followed by
45 cycles at 94°C for 45s, an appropriate annealing temperature for
45 s, and at 68°C for 1 min and 30 s. PCR products were purified using
the QIAquick system (Qiagen Ltd.). For P. concolor, approximately
650 base pairs (bp) of mtDNA (16S, ND5, and ATP8) were sequenced
(table S3).
Bear samples. Ancient DNA extracts were initially tested using
carnivore-specific primers designed to amplify a 135-bp fragment of
the hypervariable region of the mtDNA control region (39). Addition-
ally, for the ancient DNA extracts from which the 135-bp control region
fragment was successfully amplified and sequenced, a multiplex PCR
protocol (38) was applied, yielding a total of 135 to 364 bp of control
region sequence and up to 137 bp of protein-coding ATP8 sequence
and 192 bp of cytochrome b sequence (table S3). Multiplex reactions
were set up in the ancient DNA facility at ACAD. Each multiplex reac-
tion (20 ml) contained the following: 1 ml of DNA extract, 2 U of Plati-
num Taq High-Fidelity DNA Polymerase and 1× buffer (Invitrogen),
RSA (1 mg/ml; Sigma), 8 mM MgSO4, 250 mM of each dNTP, and 3 ml
of primer mix A or B (each containing the three primer pairs at a con-
centration of 1 mM). Multiplex PCR thermal cycling reactions were
conducted at 94°C for 1 min, followed by 30 cycles of denaturation at
94°C for 15 s, annealing for 20 s at 55°C, and extension at 68°C for 30 s,
followed by a final extension at 68°C for 10 min. The PCR master mix
for the singleplex reactions contained the following: 5 ml of 1:20 dilu-
tion of multiplex PCR product, 0.25 U of Platinum Taq High-Fidelity
DNA Polymerase and 1× buffer (Invitrogen), RSA (1 mg/ml; Sigma),
MgSO4, 250 mM of each dNTP, 0.75 mM forward primer, and 0.75 mM
reverse primer. Singleplex PCR thermal cycling reactions were con-
ducted at 94°C for 1 min, followed by 30 cycles of denaturation at
94°C denaturation for 15 s, annealing for 20 s at 55°C, and extension
at 68°C for 30 s, followed by a final extension at 68°C for 10 min.
Metcalf et al. Sci. Adv. 2016; 2 : e1501682 17 June 2016
Amplification products (from normal or singleplex PCRs described
above) of the expected size were purified using ExoSAP-IT or Agen-
court AMPure PCR purification kit according to the manufacturer’s
instructions.
We used Sanger sequencing methods with BigDye chemistry and
an ABI 3130xl Genetic Analyzer or an ABI PRISM capillary DNA 377
and 310 automated sequencers (Applied Biosystems) to sequence
amplicons either directly or after molecular cloning. To ensure that
our results were robust, a subset was independently replicated at the
Centre for GeoGenetics in Copenhagen, Denmark and the Henry Well-
come Ancient Biomolecules Centre in Oxford, UK. Below, we describe
independent replication for each taxon.
Camelid samples. Independent replication for successful samples
from each major taxon was performed at the Centre for GeoGenetics in
Copenhagen, Denmark. Here, the samples were drilled or crushed to
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obtain ~280 mg (10 to 660 mg) of bone powder extracted in 0.5 M
EDTA, 100 ml of proteinase K, and 0.5% SDS or N-lauryl-sarcosine at
37°C for 24 to 48 hours until all bone powder was digested. This was
followed by centrifugation using a Millipore centrifuge at 3500g for
20 to 60 min and then by DNA purification using QIAquick columns
and elution to 80 ml. Three samples were extracted in each session with
an extraction blank. Two pools of multiplex PCR consisting of eight
primers each were done as to ACAD, except 0.25 U of Taq Gold, 1× gold
buffer, 4 mM MgCl2, and BSA (1 mg/ml). The second round of single-
plex PCR was done on a 1:20 dilution of the PCR product from the first
round. Extraction blanks, and PCR controls included with every six
samples, were all blank. Sanger sequencing was done at the Macrogen
facilities (Korea), and sequences were assembled and aligned using
Sequencher version 4.8.
Phylogenetic analysis
We estimated the best nucleotide substitution model for each mito-
chondrial gene for each species by selecting the model with the lowest
Akaike’s information criterion score using ModelTest (40) or other sim-
ilar software. We inferred phylogenies using Bayesian methods in
BEAST (41) and likelihood methods using PhyML (42, 43), and reported
Bayesian prior probabilities and maximum likelihood estimates, respec-
tively, of highly supported nodes. For the closely related Puma haplo-
types, we constructed a parsimony haplotype network using TCS (44).
Details on the phylogenetic analysis of each taxon are included below.
Camelid samples. We aligned sequences by eye using Se-Al ver-
sion 2.0a11 (http://tree.bio.ed.ac.uk/software/seal/). We reconstructed
the phylogenetic relationship of camelids using Bayesian methods in
BEAST (41) and likelihood methods using PhyML (42, 43). First, we
analyzed a data set that consisted of 541 nucleotides of mitochondrial
sequence data (~432 bp of control region plus 107 bp of cytochrome b),
for a total of 78 taxa, including 30 ancient Pleistocene samples, 13 an-
cient Holocene samples, and 35 modern camelid haplotypes, which in-
clude 5 Camelus haplotypes (Fig. 2). Second, we analyzed a larger
sample set with 432 bp of mtDNA control region. These data included
88 ancient camelid samples and 77 modern camelid haplotypes (fig. S2).
We downloaded sequence data representing modern camelid genetic
diversity from GenBank (45, 46).
Felid samples. Sequences were aligned by eye using Se-Al version
2.0a11 (http://tree.bio.ed.ac.uk/software/seal/). We analyzed 343 bp
of control region and 143 bp of ATP8 mitochondrial sequence data
from P. onca mesembrina. We generated a phylogeny using a UPGMA
tree-building method with an HKY model of sequence evolution and
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RESEARCH ARTICLE
100 bootstraps (Fig. 2). We analyzed ~650 bp of 16S, ND5, and ATP8 of
sequence data from P. concolor samples, and visualized their close rela-
tionship to modern haplotypes with a parsimony haplotype network
(Fig. 2) using TCS (44).
Bear samples. We carried out phylogenetic reconstructions of
Tremarctinae bears using 694 bp of mitochondrial sequence data from
the Arctotherium sp. sample ACAD3599, along with previously
sequenced Tremarctinae bears (Tremarctos and Arctodus) and previ-
ously sequenced members of Ursine bears, the sister group to Tre-
marctinae (47–49). We used the HKY + gamma model in BEAST
1.4.8 (41) to generate a Bayesian inference phylogeny. Three inde-
pendent runs of 10 million Markov chain Monte Carlo generations
sampled every 1000 steps were done in BEAST 1.4.8 (41) using a relaxed
molecular clock (uncorrelated lognormal). The results of the three
independent runs were combined. Results of the individual and com-
bined runs were visualized using Tracer 1.4 (http://tree.bio.ed.ac.uk/
software/tracer) to check for convergence and to ensure that all Effective
Sample Size scores were >200 (suggesting sufficient sampling and run
length). The first 1 million steps (10%) of each run were discarded as
burn-in. The trees were annotated and visualized using FigTree 1.1.2
(http://tree.bio.ed.ac.uk/software/figtree).
14
C dating and calibrations
We generated 75 new date estimates of megafaunal bone and teeth
using AMS 14C dating, most of which were pretreated using ultra-
filtration to purify the gelatin and minimize contamination [following
(50)] at the Oxford Radiocarbon Accelerator Unit (ORAU) (table S1).
The 14C ages were calibrated against the SHCal13 data set (table S4)
(51). We also calibrated previously published 14C data from the large
ground sloth (M. darwinii) (14, 20, 52), the saber-toothed cat (S. popu-
lator) (22, 53), the giant short-faced bear (Arctotherium sp.) (23), and
the South American horse (H. saldiasi) (24, 25) [summarized in (4)
and (14)]. For extinct Pleistocene megafauna, the 14C ages were used
to constrain the estimates of extinction ages for megafaunal taxa using
the Phase model option in OxCal 4.2 (26) with General Outlier analysis
detection (probability = 0.05) (54). Importantly, this approach assumes
a uniform (Poisson) distribution of 14C ages to estimate the calendar age
boundaries (55); where there were less than 11 14C ages for a group, we
did not use the Phase option and instead undertook individual age cal-
ibration. Using Bayes’ theorem, the algorithms employed sample pos-
sible solutions with a posterior probability that is the product of the
prior and likelihood probabilities; the outlier option was used to detect
ages that fall outside the calibration model for each group and, if nec-
essary, down-weight their contribution to the final age estimates. Taking
into account the deposition model and the actual age measurements,
the posterior probability densities quantify the likeliest age distribu-
tions. The calibrated ages are reported in table S4. To reassure ourselves
that the Phase age modeling results are robust, we undertook individual
calibrations of the youngest samples for each Patagonian species with
greater than 11 14C dates. The ages were calibrated using SHCal13 (50) in
OxCal 4.2 (26) (table S4). All calibrated ages fall within the period of
rapid warming that followed the termination of the ACR, supporting
the approach taken here.
We used published data sets (56, 57) to estimate the arrival of humans
in Patagonia, and followed their criteria for screening radiocarbon ages.
We calibrated 14C dates using the SHCal13 data set, as described in the
previous paragraph (table S5), and estimated human arrival using the
Phase model option in OxCal 4.2 (26) with General Outlier analysis
Metcalf et al. Sci. Adv. 2016; 2 : e1501682 17 June 2016
detection (probability = 0.05) (54). A c2 test demonstrates that human
arrival was statistically different to extinction in Patagonia (df = 1, T =
17.270, 5% = 3.8).
The wide range of geographic sites, depositional situations, closely
spaced (and relatively young) 14C ages from different species, and the
statistical overlap of the Phase ages generated using OxCal 4.2 (26)
makes it highly unlikely that the narrow temporal window of the last
observations might be the result of a taphonomic or observational
artifact, such as the Signor-Lipps effect (58). Furthermore, the density
and quality of the AMS 14C dates (mostly bone or teeth samples pre-
treated using ultrafiltration at ORAU) suggest that the chronological
series are accurate. Importantly, the Signor-Lipps effect would only ex-
tend the period of apparent overlap between humans and megafauna
and further confirm our observation that the megafaunal, extinctions
occurred during the warming phase following the ACR stadial.
Downloaded from http://advances.sciencemag.org/ on June 17, 2016
We plotted the summed probabilities of the IntCal13-calibrated
(59) terminal AMS dates for North American Equus caballus, Saiga
tatarica, Panthera leo spelaea, Mammuthus primigenius, and Mammut
americanum from the study of Cooper et al. (27), and plotted these
against time. These were the only latest Pleistocene North American
megafaunal extinctions with suitably detailed 14C data sets available
(27). Timing of onset and termination of the ACR are as defined by Ant-
arctic ice core isotopic records and change in the interhemispheric radio-
carbon gradient (7, 9).
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/2/6/1501682/DC1
Supplementary Methods
Supplementary Results
fig. S1. Map of South America showing sites where genetic data were recovered.
fig. S2. Phylogeny of camelids based on 432 bp of mitochondrial control region data.
fig. S3. Mitochondrial sequence data for Arctotherium.
table S1. Fossil samples included in this study.
table S2. Samples for which DNA extraction failed and 14C failed or was not attempted.
table S3. Primer sequences used to amplify mitochondrial genes.
table S4. Megafaunal 14C ages were calibrated against the SHCal13 data set.
table S5. Published human 14C ages were calibrated against the SHCal13 data set.
table S6. Results of independent replication of ancient DNA sequences.
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Acknowledgments: We thank the following museums and universities for the samples: La Plata
Museum, La Plata Zoo, Universidad Nacional de Cuyo, Museo de Historia Natural de San Rafael,
Instituto de la Patagonia, Universidad de Magallanes, Museo de Historia Natural, Universidad Nacio-
nal Mayor de San Marcos, Zoological Museum Amsterdam, Malmo Museum of Natural History, and
Metcalf et al. Sci. Adv. 2016; 2 : e1501682 17 June 2016
Moscow State University Zoological Museum. We thank S. Silvestri, M. Reguero, E. Tonni, A. Prieto,
and J. Weinstock. We also thank the ACAD staff and researchers for their support and assistance.
Funding: This research was funded by the Australian Research Council (ARC) (DP140104233 and
DP0664562) and the UK Natural Environment Research Council (NERC) (grant NER/B/S/2003/00181
to A.C.). J.T.V. was funded by the Danish National Research Foundation (DNRF94), and R.B. was
funded by the Biotechnology and Biological Sciences Research Council (02/A1/G/08351) and NERC
(NER/B/S/2003/00181). ARC Future, Federation, and Laureate fellowships supported A.C.
(FL140100260, FT099233, and FF0457313), C.T. (FL100100195), C.J.A.B. (DP130103842 and
FT110100306), and J.J.A. (FT10010010). F.J.P. was funded by CONICET and Agencia Nacional
de Promoción Científica y Tecnológica (PICT 2011-309 and PIP 2011-164). Author contribu-
tions: A.C. conceived the project; A.C., J.L.M., C.T., R.B., S.C.B., and C.J.A.B. performed the research
and analysis; and A.C., J.L.M., and C.T. wrote the paper with input from all authors. Competing
interests: The authors declare that they have no competing interests. Data and materials
availability: The genetic sequences have been deposited in GenBank (KU753598 to
KU753727 and KU884290 to KU884323), and the new and previously published 14C data are
presented in the Supplementary Materials with references. Additional data related to this
paper may be requested from the authors.
Downloaded from http://advances.sciencemag.org/ on June 17, 2016
Submitted 21 November 2015
Accepted 27 May 2016
Published 17 June 2016
10.1126/sciadv.1501682
Citation: J. L. Metcalf, C. Turney, R. Barnett, F. Martin, S. C. Bray, J. T. Vilstrup, L. Orlando,
R. Salas-Gismondi, D. Loponte, M. Medina, M. De Nigris, T. Civalero, P. M. Fernández,
A. Gasco, V. Duran, K. L. Seymour, C. Otaola, A. Gil, R. Paunero, F. J. Prevosti,
C. J. A. Bradshaw, J. C. Wheeler, L. Borrero, J. J. Austin, A. Cooper, Synergistic roles of
climate warming and human occupation in Patagonian megafaunal extinctions during the
Last Deglaciation. Sci. Adv. 2, e1501682 (2016).
8 of 8
 Synergistic roles of climate warming and human occupation
in Patagonian megafaunal extinctions during the Last
Deglaciation
Jessica L. Metcalf, Chris Turney, Ross Barnett, Fabiana Martin,
Sarah C. Bray, Julia T. Vilstrup, Ludovic Orlando, Rodolfo
Salas-Gismondi, Daniel Loponte, Matías Medina, Mariana De
Nigris, Teresa Civalero, Pablo Marcelo Fernández, Alejandra
Gasco, Victor Duran, Kevin L. Seymour, Clara Otaola, Adolfo Gil,
Rafael Paunero, Francisco J. Prevosti, Corey J. A. Bradshaw,
Jane C. Wheeler, Luis Borrero, Jeremy J. Austin and Alan Cooper
(June 17, 2016)
Sci Adv 2016, 2:.
doi: 10.1126/sciadv.1501682

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